Myeloid-derived suppressor cells (MDSC) are immature myeloid cells described in both tumor-bearing mice and human cancer patients, which play an increasingly recognized role in cancer maintenance, progression, and resistance to immunotherapy. MDSC infiltrate solid tumors, including coetaneous melanoma, and potently inhibit anti-tumor T cell responses through a variety of mechanisms including production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS). While iNOS/NO have not previously been shown to regulate MDSC development, persistence, or localization within the tumor-bearing host, the well-known associations of iNOS with cancer, infection, shock, and other inflammatory states associated with MDSC accumulation make this an attractive hypothesis. Our preliminary data demonstrate that mice bearing syngeneic B16 or MT-RET melanomas accumulate splenic and tumor-infiltrating GR-1+CD11b+ MDSC, and experience a decline in splenic CD4+ and CD8+ T cells. Treatment with the small molecule iNOS inhibitor N6(1-iminoethyl)-L-lysine-dihydrochloride (L-NIL) decreases MDSC infiltration in tumor and spleen, reverses the tumor-mediated decrease in CD4+ and CD8+ splenocytes, and enhances the number of tumor-infiltrating CD4+ and CD8+ T cells. L-nil treatment was also associated with down regulation of activated STAT3 in the tumor, and normalization of elevated levels of VEGF and other inflammatory cytokines observed in sera of tumor-bearing mice. Serum VEGF levels were also suppressed, intratumoral MDSC infiltration reduced, and T cell numbers normalized in tumor-bearing iNOS-/- "knockout" mice, suggesting that host-derived iNOS plays a role in MDSC recruitment and migration. Together, these data suggest that MDSC recruitment and trafficking are controlled by cross-talk in which iNOS expression by tumor-infiltrating myeloid cells, melanoma cells, or both is required for release of soluble factors from the tumor which recruit additional MDSC from the bone marrow and drive their infiltration into the tumor. We further hypothesize that targeted inhibition of iNOS with small-molecule chemical antagonists will enhance the response to anti-tumor vaccination by suppressing the recruitment and suppressive capacity of intratumoral MDSC. In this proposal we aim to characterize the effect of iNOS inhibition on tumor-secreted factors responsible for recruitment and intratumoral homing of MDSC in syngeneic transplantable and spontaneous tumor models. We will seek to determine whether the effects of iNOS inhibition on production, by tumor and myeloid cells, of cytokines known to regulate MDSC are mediated by changes in STAT3 activation. We will determine whether iNOS inhibition decreases intratumoral MDSC numbers by affecting their trafficking, survival, or differentiation, or through a combination of these mechanisms. Finally, we will use a well-established model of adoptive CD8+ T cell transfer and anti-tumor vaccination to determine whether targeted iNOS inhibition boosts the number, effector function, and anti-tumor efficacy of vaccine-induced CD8+ CTL. The above project will be carried out in the context of a comprehensive, mentored career development plan leading to scientific independence. A team of outstanding mentors will provide critical guidance in every aspect of the candidate's scientific development, from hands-on critique of experimental design and data analysis, to publication of results, to acquisition of lab management and grant-writing skills which will lead to independent funding. These personal interactions will be supplemented by formal class work addressing specific areas targeted for improvement (biostatistics, clinical research skills and translation of basic findings to clinical trials). As the candidate is a physician-scientist with access to unique patient populations and clinical material, special emphasis has been placed on providing didactic background and supplemental mentorship in clinical research. The candidate has the benefit of a first class research environment in which immunology and cancer biology are centers of excellence within the institution, and already has secured the confidence and support of his department and the institution. His immediate goal of carrying out and publishing the research proposed in this application will significantly enhance our understanding of the relationship between inflammation and tumor-mediated immunosuppression, and provide a sound platform for establishing an independent research career in cancer immunology and translational cancer biology. PUBLIC HEALTH RELEVANCE: Once mechanism used by cancers to shield themselves from immune responses is to induce the generation of so-called "myeloid-derived suppressor cells" (MDSC) which are white blood cells that suppress anti-tumor T cell responses. We have discovered that, by blocking the activity or expression of one of the body's inflammatory molecules - the enzyme inducible nitric oxide synthase (iNOS), it is possible to inhibit the accumulation and immune-suppressing activity of MDSC in a mouse model of melanoma (skin cancer);these finding s may ultimately be used to improve cancer immunotherapy. The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the "Critique" section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The "Resume and Summary of Discussion" section above summarizes the final opinions of the committee.